Typically, such an optical apparatus includes a junction device for the beam path, for example a beam splitter. The junction device is located between the light source of the laser beam and the sample. Consequently, the fluorescence light emanating from the sample is deflected laterally away from the direction of the excited laser beam and towards the photo sensor.
A scanning microscope is known from Nature, Vol. 183, page 760. Several partial beams are formed from an expanded laser beam. The partial beams are focused by a common objective lens to optically excite a sample. A photo sensor arranged behind the objective lens, as seen from the sample, registers fluorescence light of the focal points which emanates from the sample. Several partial beams are formed from the laser beam by a screen or an aperture arrangement having several holes. The screen or aperture arrangement is arranged in the optical path of the apparatus, so that the fluorescence light coming from the sample has to pass through the screen when it is to contact the photo sensor. Consequently, the apparatus is a confocal scanning microscope in which only fluorescence light coming from one plane of the sample contacts the photo sensor. Thus, a three-dimensional resolution of the sample with respect to the attained fluorescence light intensities is possible. The screen or aperture arrangement of the scanning microscope is a so-called "Nipkow disc" having holes arranged therein to scan the sample uniformly by rotating the "Nipkow disk" about its center. The "Nipkow disk" was originally designed to scan pictures to attain a signal to be send by telegraph.
A problem of the known scanning microscope is that the holes of the blind must have a certain minimum lateral distance between one another to prevent fluorescence light emanating from a plane of the sample other than the plane to be observed and not being focused back to the origin hole of the exciting radiation from passing through holes adjacent to the origin hole and onto the photo sensor. A great distance between the holes within the blind results in the luminous power of the laser beam substantially fading out due to the aperture arrangement and consequently not being used. Additionally, the laser light faded out by the aperture arrangement is reflected onto the photo sensor from the rear side of the aperture arrangement and causes great background.
To solve this problem, it is suggested in Nature, Vol. 338, pages 804 through 806, to arrange the holes of the aperture arrangement especially close to one another, knowing that light unfocused with respect to the origin hole passes through adjacent holes of the aperture arrangement and onto the photo sensor. The light resulting from the background is to be compensated by considering a light intensity distribution recorded by a photo sensor prior to the compensation and without making use of the aperture arrangement. After that, the light intensity distribution recorded without the aperture arrangement is subtracted from light intensity distribution recorded using the aperture arrangement. This procedure may cause difficulty since the background to be subtracted may have a greater dimension than the signal to be observed. Consequently, extreme defects may occur in the revised signal.
A scanning microscope is known from U.S. Pat. No. 5,034,613. Fluorescence light of the sample is registered by the photo sensor. The fluorescence light has a wavelength which is half as long as the wavelength of the laser beam. The fluorescence of the sample is based on a two photons excitation. The probability of such a two photons excitation differs substantially from zero exclusively in the core region of each focal point in which the laser beam is focused for optical excitation of the sample. Thus, the scanning microscope has a substantially improved axial resolution compared to confocal scanning microscopes. Nevertheless, the yields of fluorescence light of the sample are relatively smaller compared to a confocal scanning microscope. Thus, measuring times necessary for each sample to attain meaningful fluorescence light intensities are increased. The scanning of a sample in all three dimensions takes a much longer period of time compared to a confocal scanning microscope. Furthermore, the laser beam of the known scanning microscope making use of the two photons excitation is only focused in one focal point to excite the sample. The yield of fluorescence light of this focal point can be increased by increasing the luminous power of the laser beam. However, the possibility of increasing the luminous power of the laser beam is strictly limited since otherwise a local change of the sample occurs due to overheating.
From the German Patent Application 40 40 441 another scanning microscope is known. A laser beam is divided into two portions, and the two portions coming from opposite directions are brought to interference in a common focal point to excite a sample. With the interference, a main maximum and two secondary maxima of the light intensity occur in the region of the common focal point of the two partial beams. The main maximum has a smaller axial extension and is easily separable from the secondary maxima by a confocal arrangement. The small axial extension of the main maximum implies a very good axial resolution of this known scanning microscope.
A scanning microscope of the type mentioned at the beginning is known from Bioimages 4 (2): 57-62, June 1996. The micro lenses are arranged to form a micro lens wheel. The laser beam is divided into partial beams, and the sample is scanned in two dimension by rotating the micro lens wheel about its axis extending in parallel to the laser beam. A aperture wheel including one aperture opening for each micro lens is arranged behind the micro lens wheel. The partial beam directed onto the sample, as well as the fluorescence light excited by this partial beam and coming from the direction of the sample, enter through the aperture openings to attain a sufficient resolution in depth for this known scanning microscope using one photon excitation. The arrangement and the support of the micro lens wheel and of the aperture wheel have to be executed extremely accurate to assure a perfect function of the known scanning microscope. Since the micro lenses in the known micro lens wheel are not arranged side by side in two dimension, but in bent rows instead, as it is realized in a usual "Nipkow disk", not the entire light intensity of the expanded laser beam is used.